Mosquito (Diptera: Culicidae) assemblages associated with Nidularium and Vriesea bromeliads in Serra do Mar, Atlantic Forest, Brazil

R E S E A R C H

Open Access

Mosquito (Diptera: Culicidae) assemblages

associated with

Nidularium

and

Vriesea

bromeliads in Serra do Mar, Atlantic Forest, Brazil

Tatiani C Marques, Brian P Bourke, Gabriel Z Laporta and Maria Anice Mureb Sallum

Abstract

Background:The most substantial and best preserved area of Atlantic Forest is within the biogeographical sub-region of Serra do Mar. The topographic complexity of the region creates a diverse array of microclimates, which can affect species distribution and diversity inside the forest. Given that Atlantic Forest includes highly heterogeneous

environments, a diverse and medically important Culicidae assemblage, and possible species co-occurrence, we evaluated mosquito assemblages from bromeliad phytotelmata in Serra do Mar (southeastern Brazil).

Methods:Larvae and pupae were collected monthly fromNidulariumandVrieseabromeliads between July 2008 and June 2009. Collection sites were divided into landscape categories (lowland, hillslope and hilltop) based on elevation and slope. Correlations between bromeliad mosquito assemblage and environmental variables were assessed using multivariate redundancy analysis. Differences in species diversity between bromeliads within each category of elevation were explored using the Renyi diversity index. Univariate binary logistic regression analyses were used to assess species co-occurrence.

Results:A total of 2,024 mosquitoes belonging to 22 species were collected. Landscape categories (pseudo-F value = 1.89, p = 0.04), bromeliad water volume (pseudo-F = 2.99, p = 0.03) and bromeliad fullness (Pseudo-F = 4.47, p < 0.01) influenced mosquito assemblage structure. Renyi diversity index show that lowland possesses the highest diversity indices. The presence ofAn. homunculuswas associated withCx. ocellatusand the presence ofAn. cruziiwas associated withCx. neglectus, Cx. inimitabilis fuscatusandCx. worontzowi. Anopheles cruziiandAn. homunculuswere taken from the same bromeliad, however, the co-occurrence between those two species was not statistically significant.

Conclusions:One of the main findings of our study was that differences in species among mosquito assemblages were influenced by landscape characteristics. The bromeliad factor that influenced mosquito abundance and assemblage structure was fullness. The findings of the current study raise important questions about the role ofAn. homunculusin the transmission ofPlasmodiumin Serra do Mar, southeastern Atlantic Forest.

Keywords:Culicidae,Vriesea, Nidularium, bromeliads, species co-occurrence,Anopheles cruzii, Anopheles homunculus, Plasmodiumvectors, Atlantic Forest

Background

The tropical forest of eastern South America, known as Atlantic Forest, is one of the world’s most important biodiversity hotspots [1]. The most substantial and best preserved area of Atlantic Forest is found in the biogeo-graphical sub-region of Serra do Mar [2], which contains approximately 30% of the remaining forest [3]. Although

climatic conditions are relatively uniform across Serra do Mar, the topographic complexity of the region [4] creates a diverse array of microclimates. These topogra-phical differences provide gradients of oxygen, humidity and temperature that can affect species distribution and diversity inside the forest [5,6]. As such, topography is likely to be an important factor in shaping floral [4] and faunal [6] diversity in Atlantic Forest.

A rich diversity of species from the Culicidae family is found in Serra do Mar [7,8]. Many of these mosquito

* Correspondence: [email protected]

Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, Av. Dr. Arnaldo 715, CEP 01246-904, São Paulo-SP, Brasil

species appear to have evolved in close association with

bromeliads [9]. For example,Culex ocellatusTheobald

and many species of Culex (Microculex) Theobald

[10,11] and the subgenera Hystatomyia Dyar and

PhoniomyiaTheobald of the genusWyeomyiaare highly dependent upon bromeliads for larval habitat [9]. With

the exception of Anopheles (Kerteszia)bambusicolus

Komp, which is associated with bamboo internodes, the larva and the pupa ofKertesziaTheobald species depend on bromeliad phytotelmata from preserved environ-ments as their primary larval habitat [12]. The larvae of

Anopheles (Kerteszia) cruzii Dyar & Knab, Anopheles

(Kerteszia)bellatorDyar & Knab, andAnopheles( Kertes-zia)homunculusKomp are frequently found in Nidular-iumandVrieseabromeliads in Atlantic Forest [13].

Human malaria is endemic in Serra do Mar [14] where the primary vectors areAn. cruziiandAn. bellator[15].

Anopheles homunculushas also been incriminated as a

vector of humanPlasmodium parasites in Paraná and

Santa Catarina states [16]. Despite the medical importance of these three species, many aspects of their biology are poorly known [15]. Consequently, studying the way in which environmental variables influence the presence and distribution of these species may help to determine the role of vectors in the dynamics of human plasmodium transmission in Serra do Mar.

Considering the existence of environmental determi-nants for mosquito assemblages in larval habitats [17], and a highly heterogeneous environment [4] and diverse medically important Culicidae assemblage in Serra do Mar [7], the main objectives of the study are to: (1) char-acterize bromeliad mosquito assemblage structure; (2) assess correlations between bromeliad mosquito assem-blage structure and various environmental factors; (3) determine correlations between the most abundant spe-cies and various bromeliad characteristics; and (4) assess co-occurrence among species.

Methods

The study area is located in the Aroeira District (25° 0’ 54"S and 47° 55’37"W, SAD 69) of Cananéia, Serra do Mar, São Paulo state (Figure 1). Collection sites were divided into three distinct landscape categories based on elevation, slope and site accessibility. They were: 1) low-land (5-20 m altitude), 2) hillslope (33 to 54 m altitude), and 3) hilltop (81 to 263 m altitude). All three sites were of primary and considerably preserved forest. The lowland area was found adjacent to an estuarine channel and man-grove. The area was very humid, with a high tree density and little light penetration at ground level. Rocky outcrops were scattered in this area, but abundant at higher eleva-tions (hillslope and hilltop), which may have lowered humidity and increased light penetration at the hilltop.

Larvae and pupae were collected from terrestrial, epi-phytic and saxicolousNidularium(Subfamily: Bromelioi-deae) andVriesea(Subfamily: Tillandsioideae) bromeliads in each of the three elevation categories at monthly inter-vals from July 2008 until June 2009. This yielded samples from 36 plants in each category, totaling 108 bromeliads. Monthly sampling sites varied spatially (by approximately 500 m) in order to increase the coverage area. Of the 108 bromeliads sampled, 94 wereNidulariumand 14Vriesea. Plants of both genera were equally sampled among

eleva-tion categories. Thirty Nidularium bromeliads were

sampled on the lowland, 33 on the hillslope and 31 on the hilltop whilst 6Vrieseawere sampled on the lowland, 3 on the hillslope and 5 on the hilltop.

Data taken for each sampled plant were: type of brome-liad (terrestrial, epiphytic and saxicolous), bromebrome-liad height, water pH, tank diameter, tank depth, and water volume. Water from each bromeliad was removed with a manual suction pump and measured for the water volume. Additional fresh water was poured into each plant and the removal was again repeated. Samples from each bromeliad were kept in separate plastic containers and the suction pump was washed with both 70% ethanol and fresh water to avoid cross contamination between plants. Climatic data from the two weeks preceding the collection date were obtained from the Centro Integrado de Informações Agrometeorológicas - CIIAGRO (http://www.ciiagro.sp. gov.br/) station in Cananéia.

Larvae and pupae were taken to the laboratory and raised to adulthood for species identification. Larval and pupal exuviae were mounted on microscopy slides with Canada balsam. Whenever possible, male genitalia were used for species identification. Morphological identifica-tion was based on Lane and Whitman [18], Lane [19], Correa and Ramalho [20], Cotrim and Galati [21] and For-attini [22]. Nine specimens that could not be identified using Lane’s [19] keys were registered asCx. (Mcx.) sp1 according to Marques’et al. [11] informal nomenclature. For the purpose of the present study, we identified larvae with evident subapical swelling asCx. daumasturus[23], a species that was formerly synonymized withCx. imitator imitator[19]. However, the validation of the species needs further investigation. Identification ofAn. cruziiandAn. homunculuswas based on characters of the fourth-instar larva, pupa and adults following the characteristics pro-posed by Forattini [22] and Sallum et al. [24]. Adults asso-ciated with either larval or pupal exuviae were deposited in Coleção Entomológica de Referência da Faculdade de Saúde Pública da Universidade de São Paulo (FSP-USP).

[28] in order to assess the sampling effort. This estimator performs best with respect to accuracy in both even and uneven communities [29]. The multivariate redundancy analysis (RDA) [30] was used to assess correlations between bromeliad mosquito assemblage structure and various environmental variables. These variables included collection site landscape category (m), mean temperature (°C), total precipitation of rain two weeks prior to collec-tion (mm), type of bromeliad (terrestrial, epiphytic and saxicolous), bromeliad height from the ground (m), tank diameter (cm), tank depth (cm), water pH, bromeliad water volume (ml), and an estimate of bromeliad tank fullness (ml/cm; water volume divided by tank height).

The Renyi diversity index [31] was used to explore dif-ferences in species diversity among bromeliads within each category of elevation (lowland, hillslope, and hill-top). This index provided four further diversity indices: Total richness, Shannon-Weiner index, Simpson-Yule index, and the Berger-Parker index. The differences

between landscape categories for each of these indices were then tested for statistical significance using Krus-kal-Wallis test (p < 0.05).

Correlations between species abundance and both bromeliad fullness and landscape category were assessed based on the results from the RDA analysis. Univariate Gaussian regression analyses were per-formed to determine whether the bromeliad fullness had either a positive or negative contribution to species abundance (n = 108, p < 0.05). Univariate binomial negative regression analyses were then carried out for landscape categories (n = 108). Relative species abun-dance (prevalence ratio) values for hillslope and hilltop were estimated using the lowland category as the base-line. A prevalence ratio of one indicates the given spe-cies is not associated with any landscape category, whereas values of greater than one and less than one indicate a positive and negative association, respectively (p < 0.05, CI 95%).

Univariate binary logistic regression analyses (n = 108) were used to assess species co-occurrence in bromeliads. Species abundance was transformed into dummy variables (absence = 0, presence = 1), and the analyses of these vari-ables provided one of three possible results; an odds ratio value of one indicates species are associated randomly, whereas odds ratio values of greater than one and less than one indicate a positive and negative association, respectively (p < 0.05, CI 95%).

Results

Two thousand and twenty four mosquitoes belonging to 22 species were collected (Table 1).Culex ocellatus(429; 21.20%),Cx. (Mcx.)imitator retrosus(388; 19.17%),Cx. (Mcx.)neglectus(350; 17.29%),Cx. (Mcx.)imitator imita-tor(213; 10.52%),An. homunculus(201; 9.93%),Cx. (Mcx.)

inimitabilis fuscatus(126; 6.23%),An. cruzii(106; 5.24%),

Cx. (Mcx.)worontozowi(80; 3.95%) andCx. (Mcx.) aphy-lactus(55; 2.52%) were the most abundant species.

The extrapolated species richness using the second-order jackknife estimator was 35 species. Of the 22 species collected, two were found to be singletons and four dou-bletons. Results of the RDA showed that landscape

categories, bromeliad water volume and bromeliad fullness had a significant influence on the bromeliad mosquito assemblage structure (pseudo-F value = 1.89, p = 0.04; pseudo-F value = 2.99, p = 0.03; and pseudo-F value = 4.47, p < 0.01, respectively; Table 2).

The Renyi diversity curves showed that the lowland possesses the highest diversity indices, and that the values for the hillslope are more similar to those obtained for the hilltop (Figure 2; Additional file 1). The Kruskal-Wallis tests indicated significant difference in the Total richness (= 0) and the Shannon-Weiner diversity index

(= 1; Figure 2) between lowland and hilltop (KWc2=

4.90, p = 0.03 and KWc2 = 5.75, p = 0.02, respectively). Further results are included in Additional file 1.

Univariate Gaussian linear regression analyses found that bromeliad fullness was positively associated with

Cx. imitator imitator (b1= 0.90; p < 0.001),An. cruzii(b 1= 0.19; p < 0.01),Cx. neglectus (b 1= 0.68; p < 0.01) andCx. aphylactus (b1= 0.10; p = 0.03).Cx. inimitabilis fuscatus, however, showed a negative association (b1= -0.32; p = 0.04) indicating this species was more asso-ciated with shallow water inside bromeliad tanks (Addi-tional file 2).

Table 1 Species of Culicidae found inNidulariumandVrieseabromeliads.

Species Landscape cateogories Total

Lowland Hillslope Hilltop

Anopheles(Kerteszia)cruziiDyar & Knab 43 24 39 106

Anopheles(Kerteszia)homunculusKomp 98 62 41 201

Culex ocellatusTheobald 101 114 214 429

Culex(Microculex)sp1 7 2 0 9

Culex(Microculex)reducensLane & Whitman 0 1 5 6

Culex(Microculex)worontzowiPessoa & Galvão 11 10 59 80

Culex(Microculex)daumasturus(Kum) 4 0 1 5

Culex(Microculex)imitator imitatorTheobald 165 35 13 213

Culex (Microculex) imitator retrosus Lane & Whitman 81 159 148 388

Culex(Microculex)aphylactusRoot 20 20 15 55

Culex(Microculex)inimitabilis fuscatusLane & Whitman 29 53 44 126

Culex(Microculex)microphyllusRoot 0 4 14 18

Culex(Microculex)neglectusLutz 199 113 38 350

Culex(Microculex)intermediusLane & Whitman 8 0 0 8

Culex(Microculex)pleuristriatusTheobald 2 0 0 2

Runchomyia(Runchomyia)theobaldi(Lane & Cerqueira) 0 1 0 1

Wyeomyia(Phoniomyia)davisi(Lane & Cerqueira) 2 0 0 2

Wyeomyia(Phoniomyia)galvaoi(Correa & Ramalho) 4 3 0 7

Wyeomyia(Phoniomyia)incaudata(Root) 2 0 0 2

Wyeomyia(Phoniomyia)palmata(Lane & Cerqueira) 2 0 0 2

Wyeomyia(Phoniomyia)pilicaudaRoot 0 0 1 1

Wyeomyia(Phoniomyia)theobaldi(Lane & Cerqueira) 8 5 0 13

Total abundance 786 606 632 2024

Total species richness 18 15 13 22

Univariate negative binomial regression analyses found thatAn. homunculus(prevalence ratio = 0.42; CI 95% = 0.18 - 0.95) andCx. neglectus(prevalence ratio = 0.19; CI 95% = 0.07 - 0.5) were more closely associated with

low-land than hilltop, and thatCx. imitator imitatorwas

more closely associated with lowland than either hilltop (prevalence ratio = 0.08; CI 95% = 0.03 - 0.22) or hillslope

(prevalence ratio = 0.21; CI 95% = 0.08 - 0.53) (Addi-tional file 3).

Univariate binary logistic regression analyses for

co-occurrence showed thatAn. homunculuswas associated

withCx. ocellatusandAn. cruziiwas associated withCx. neglectus, Cx. inimitabilis fuscatusandCx. worontzowi

(Table 3). AlthoughAn. cruziiandAn. homunculuswere

Table 2 Correlation between bromeliad mosquito assemblage and environmental variables.

Environmental variable Type of variable Redundancy analysis

Mean temperature Numeric, from 16.9 to 27.5°C Pseudo-F = 0.69, d.f. = 106 (p = 0.63) Total precipitation Numeric, from 0.9 to 155.3 mm Pseudo-F = 0.13, d.f. = 106 (p = 0.99) Bromeliad height Numeric, from 0 to 260 cm Pseudo-F = 0.61, d.f. = 106 (p = 0.68) Type of bromeliad Factor, 3 levels (ground, epiphytic, rocky) Pseudo-F = 1.49, d.f. = 106 (p = 0.13)

Water pH Numeric, from 3.9 to 7.5 Pseudo-F = 0.41, d.f. = 106 (p = 0.86)

Tank diameter Numeric, from 10 to 100 cm Pseudo-F = 0.50, d.f. = 106 (p = 0.75) Tank depth Numeric, from 10 to 140 cm Pseudo-F = 0.98, d.f. = 106 (p = 0.41) Landscape categories Factor, 3 levels (lowland, hillslope, hillside) Pseudo-F = 1.89, d.f. = 106 (p = 0.04)a Bromeliad water volume Numeric, from 7 to 750 ml Pseudo-F = 2.99, d.f. = 106 (p = 0.03)a Bromeliad fullness (water divided by depth) Numeric, from 0.2 to 17.5 ml/cm Pseudo-F = 4.47, d.f. = 106 (p < 0.01)a

Results of the multivariate Redundancy analysis (RDA) showing correlations between bromeliad mosquito assemblage structure and environmental variables. a = Significant result under the null hypothesis: Pseudo-F = 0 (p < 0.05).

occasionally found in the same bromeliad, this associa-tion was not statistically significant (1.82, p = 0.14). Con-sidering mosquito frequency distribution among bromeliad genera (Additional file 4), results of co-occur-rence analyses were mainly because ofNidularium. The

number of plants sampled of theNidulariumgenus was

approximately seven times higher than those of the Vrie-sea(94Nidulariumversus 14Vriesea).

Discussion and conclusions

The difference between the observed (n = 22) and extrapo-lated species richness (n = 35) in our study can be explained by the high number of rare species present in the tropical forest of Serra do Mar [4]. According to the second order jackknife estimator, we detected six rare spe-cies in our study, which constitutes approximately one quarter of the observed species. It therefore appears that rarer species were under-represented in our study, which may have affected our ability to detect the significance of some determinants (Table 2). However, those determi-nants that were significant for the assemblage structure were also significant for the most abundant species.

Alves et al. [4] found significant differences in forest structure and biomass variation along a 0-1100 m alti-tude gradient of coastal Atlantic Forest in Serra do Mar. The authors also showed how small changes in elevation in tropical regions can significantly affect various envir-onmental factors such as air temperature, solar radiation, light availability, edaphic discontinuities, both soil moist-ure and temperatmoist-ure, nutrients availability, ground eva-poration and microbial decomposition. Considering that environmental variables may have an impact on the avail-ability and suitavail-ability of mosquito habitats, including lar-val habitats (e.g. bromeliads), one should expect to find differences in Culicidae assemblages in different locations within Atlantic Forest. In the present study, differences in mosquito community structure in a small gradient of alti-tude (0-263 m) were found, showing that the complexity of Serra do Mar and elevational gradient may be consid-ered in ecological studies of Culicidae. This finding is

consistent with that of Navarro et al. [32] who found that elevation is an important landscape determinant for Culi-cidae fauna distribution in Venezuela.

One of the main findings in our study was that differ-ences in species among mosquito assemblages were influ-enced by elevation categories. The higher total species richness found in the lowland (Table 1) is consistent with greater niche availability, and may be related to higher spe-cies abundance because of the decreased probability of local population extinction [33]. Micro-climatic variation could not be evaluated because such data was not available but is likely to be important in influencing mosquito fauna and their distribution. The macro-climatic data that was used in our study could not explain differences in assem-blage structure (Table 2).

Previous studies showed that differences in species among mosquito assemblages can be explained by brome-liad characteristics [34,35]. The quantity and quality of food resources, and physico-chemical properties of the water in bromeliads tanks may determine the species found in them [34]. It is noteworthy that pH, conductivity,

temperature, and O2concentration were found unrelated

to both richness and species diversity of macro-invertebrate fauna inhabitingTillandsia turneriBaker (Bromeliaces) in high altitude forest in Colombia [36]. However, bromeliad water volume and plant area were associated with abun-dance. Machado-Allison et al. [37] found a positive correla-tion between bromeliad structural complexity, habitat persistence, presence of predators and mosquito species richness. In another study, Araújo et al. [38] found that species abundance was positively associated with increased water volume, whereas richness was correlated with plant diameter. In considering that larval mosquito community structure may be influenced by both the volume of water inside a bromeliad tank and depth of the water, another variable named bromeliad fullness was assessed forVriesea

andNidulariumplants from Atlantic Forest. This variable was found to have a statistically significant influence on the mosquito community structure. Water fullness may create an array of micro-variation in the physico-chemical Table 3 Co-occurrence between mosquito species inNidulariumandVrieseabromeliads.

Species Odds ratio (95%CI),

Anopheles cruzii Odds ratio (95%CI),Anopheles homunculus

Anopheles homunculus 1.82 (0.83, 4.01)

-Culex ocellatus 0.84 (0.36, 1.95) 2.43 (1.06, 5.56)a

Culex aphylactus 1.68 (0.62, 4.58) 0.96 (0.36, 2.6)

Culex imitator imitator 1.22 (0.55, 2.73) 1.55 (0.71, 3.4)

Culex imitator retrosus 1.52 (0.69, 3.34) 1.23 (0.57, 2.64)

Culex inimitabilis fuscatus 3.73 (1.33, 10.5)a _{2.1 (0.76, 5.83)}

Culex neglectus 2.87 (1.28, 6.43)a _{0.99 (0.46, 2.11)}

Culex worontzowi 4.65 (1.33, 16.27)a _{0.91 (0.29, 2.92)}

characteristics of water content, and thus in food resources and presence of predators that can affect mosquito larval community structure.

Mosquito species abundance may also be influenced by bromeliad taxa [9,39-42]. Navarro et al. [43] found mos-quito taxa association with bromeliad family in Venezuela. Similarly, in Panaquire, Venezuela, Machado-Allison et al. [44] found that some species of mosquitoes were strongly associated with species of bromeliads. In the present study, among the six rarest mosquito species found in

NidulariumandVrieseabromeliads, four belonged to the

Wyeomyia(Phoniomyia). Similarly, Müller and Marcondes [45] collected only a single individual ofWyeomyia( Pho-niomyia) in plants ofNidularium innocentii. However, Mocellin et al. [46] foundWyeomyia(Phoniomyia) to be the most abundant taxa inNeoregelia compacta(Mez) and

Billbergia nanaE. Pereira in the Botanical Garden of Rio de Janeiro. It therefore appears that plants of the genus

Nidulariumdo not represent important larval habitat for

Wyeomyia (Phoniomyia). Given the large numbers of

adult Wyeomyia(Phoniomyia) previously found in the

study area [47], it is likely that this subgenus favors alter-native bromeliad genera, for example,Neoregeliaand Bill-bergia. Moreover, it is noteworthy that at least 21 additional genera of Bromeliaceae can be found in Serra do Mar [48], some of which may provide important larval habitat forWyeomyia(Phoniomyia).

The subgenusKertesziais comprised of 12 species, of which four have been implicated as malaria vectors [12]. Three of these,An. bellator, An. cruzii, andAn. homuncu-lus, are important vectors in the Atlantic Forest. While

An. bellatorandAn. cruziiare widely distributed, the geo-graphical distribution ofAn. homunculusis poorly known [15]. Sallum et al. [24] stated that the lack ofAn. homun-culusrecords in Brazil may be a consequence of an inabil-ity to effectively identify the species based on female morphological characters. Larval and pupal characteristics, on the other hand, are highly effective at resolving this species. Despite the abundance ofAn. cruziiandAn. bella-torfound in the study area by Forattini et al. [49], we only identifiedAn. cruziiandAn. homunculus. The absence of

An. bellatormay be because of a preference for larval habi-tat either in the canopy [13] or restinga [50], but plants

from both environments were not sampled.Anopheles

homunculus, on the other hand, was frequently encoun-tered in our study and made up approximately 65% of all

Kertesziaspecimens collected.Anopheles homunculuswas similarly found to be common in enclosed, humid forests in Serra do Mar in Santa Catarina state [50]. It is note-worthy that Harbach and Navarro [51] reported

An. homunculusin forests at altitudes of up to 1700 m in Auyantepui, Venezuela.

Great importance lies in the ability to effectively identify the presence of malaria vectors, and understand

mechanisms involved in multiple species coexistence. The identification of a surrogate species may be used as a tool to assess the presence of vector species. In our study area, three species from the genusCulexwere found to be indi-cative of the presence ofAn. cruziiand one was found to

be indicative ofAn. homunculus(Table 3), and mostly

withinNidulariumplants (Additional file 4). However, the field observations conducted for the present study do not allow addressing ecological and evolutionary mechanisms of mosquito species coexistence. Consequently, hypotheses of species co-occurrence need further investigations employing an experimental design that combines field investigation and also mathematical modeling.

There are few published accounts ofAn. homunculusin the Atlantic Forest, and this study is the first to include this species in an assessment of diversity among mosquito assemblages.Anopheles homunculusseems to be abundant inNidulariumandVrieseabromeliads, sharing the same plants withAn. cruzii, and presence of both species were found to be associated with distinct categories of elevation and slope. The findings of the current study open

ques-tions about the importance of An. homunculusin the

transmission ofPlasmodiumsp. in Atlantic Forest. The

species was incriminated as a vector on coastal areas of Santa Catarina state [16], but its current role in human malaria transmission is largely unknown. It is noteworthy thatAlouattamonkeys were found infected with Plasmo-dium vivax, P. malariaeandP. falciparumin Serra do Mar, and thus they may act as reservoirs for human Plas-modium[52,53]. In considering thatAn. homunculusis found in areas with dense forest coverage [50] where

Alouattamonkeys are present, it is plausible to propose that either the species, or otherKertesziaspecies, may be involved in the transmission of malaria parasites from

monkeys to humans. The involvement ofAn. homunculus

in the dynamics of malaria transmission in Atlantic Forest requires further investigation, with a particular emphasis on greater sampling and testing for infectivity across the region in different levels within the forest.

Additional material

Additional file 1: Differences between landscape categories for each ofavalue of the Renyi index tested for statistical significance using Kruskal-Wallis test. Results of Kruskal-Wallis test to assess statistical significance of Renyi index values.

Additional file 2: Results of univariate Gaussian regression analyses performed with species abundance against the bromeliad fullness (volume of water divided by depth of bromeliad tank). Results of univariate regression analysis to determine correlation between species abundance and bromeliad fulness defined as volume of water divided by depth of the bromeliad tank.

regression analysis showing correlations between species abundance and elevation.

Additional file 4:Species of Culicidae taken from the genera Nidularium and Vriesea. *Number of plants in which a species was found/number of plants sampled in a specific landscape category. List of species of Culicidae collected from Nidularium and Vriesea showing the number of plants in which a species was found and the number of plants sampled in a specific landscape category.

Acknowledgements and funding

We are in debt to the reviewers for suggestions and comments that greatly improved the first draft of the manuscript; to A. Fernandes for assistance in the specimens identification; to field team from Departamento de Epidemiologia for helping with field collections. To Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (Processo n° 05/53973-0, and 2011/22088-1), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq BPP 300351/2008-9) for financial support. TCM was a recepient of CNPq fellowship no. 136557/2008-2e.

Authors_’contributions

TCM and MAMS conceived and designed the experiments. TCM did the field collections and identified the specimens. GZL performed the statistical analysis in the R package with contributions by BPB, MAMS and TCM. MAMS, TCM, BPB, GZL wrote the paper. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Received: 4 August 2011 Accepted: 16 February 2012 Published: 16 February 2012

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doi:10.1186/1756-3305-5-41

Cite this article as:Marqueset al.:Mosquito (Diptera: Culicidae) assemblages associated withNidulariumandVrieseabromeliads in Serra do Mar, Atlantic Forest, Brazil.Parasites & Vectors20125:41.

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